1. We examined whether pituitary adenylate cyclase-activating polypeptide with 38 or 27 residues (PACAP_38 or PACAP_27) serves as an intra-islet regulator of glucose-induced insulin secretion in rats. PACAP antiserum specific for PACAP_38 and PACAP_27 was used to neutralize the effect of endogenous PACAP in islets. PACAP release from islets was bioassayed using the response of cytosolic Ca¥ concentration ([Ca¥]é) in single â-cells, monitored by dual-wavelength fura_2 microfluorometry. Expression of PACAP mRNA was studied by reverse transcription-polymerase chain reaction (RT_PCR), while expression of PACAP was studied by metabolic labelling and immunoblotting. Localization of PACAP receptors was studied immunohistochemically. 2. High glucose-stimulated insulin release from isolated islets was attenuated by PACAP antiserum but not by non-immune sera. 3. The islet incubation medium with high glucose (Med) possessed a capacity, which was neutralized by PACAP antiserum, to increase [Ca¥]é in â_cells. PACAP antiserum also neutralized the [Ca¥]é-increasing action of synthetic PACAP_38 and PACAP_27, but not that of vasoactive intestinal polypeptide (VIP) and glucagon. 4. Both Med and synthetic PACAP increased [Ca¥]é in â_cells only in the presence of stimulatory, but not basal, glucose concentrations. In contrast, ATP, a substance that is known to be released from â_cells, increased [Ca¥]é in â_cells at both basal and stimulatory glucose concentrations. 5. Expression of PACAP mRNA and biosynthesis of PACAP_38 were detected in islets and a â_cell line, MIN6. 6. Immunoreactivity for PACAP-selective type-I receptor was observed in islets. 7.[Ca¥]é measurements combined with immunocytochemistry with insulin antiserum revealed a substantial population of glucose-unresponsive â_cells, many of which were recruited by PACAP_38 into [Ca¥]é responses. 8. These results indicate that PACAP_38 is a novel islet substance that is synthesized and released by islet cells and then, in an autocrine andÏor paracrine manner, potentiates and arouses â-cell responses to glucose, thereby amplifying glucose-induced insulin secretion in islets.
In our study, 5'-nucleotidase was released from bovine liver by the treatment with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and purified to a homogeneous state by concanavalin A-Sepharose and (diethylaminoethyl)-Toyopearl column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified 5'-nucleotidase were then cleaved by cyanogen bromide (CNBr), and then inositol phosphoglycan-containing C-terminal peptides (IPG peptides) were separated by C18 reverse-phase liquid chromatography and analyzed by peptide sequencer, amino acid analyzer, gas chromatography (GC), and GC-mass spectrometry (MS). Ser523 of the amino acid sequence deduced from 5'-nucleotidase cDNA [Suzuki et al. (1993) J. Biochem. (Tokyo) 113, 607-613] is revealed to be the C-terminal amino acid to which a glycosylphosphatidylinositol is anchored. Separated peaks of CNBr-cleaved IPG peptides were then analyzed by electron spray ionization (ESI)-MS. Eight different molecular weight (MW) species of CNBr-cleaved IPG peptides were detected. Three fractions of CNBr-cleaved IPG peptides were separately treated by trypsin, and trypsinized IPG peptides were purified by C18 reverse-phase liquid chromatography. Finally, five different MW species of trypsinized IPG peptides (1629.4, 1752.7, 1791.8, 1832.8, and 1994.5) were detected by ESI-MS. Together with sequential exoglycosidase treatment and quantitative analysis of sugar moieties by GC and GC-MS, microheterogeneity in the structures of these five glycosylphosphatidylinositol (GPI) anchor species was determined. The common core structure was ethanolamine phosphate-mannose-mannose-mannose(-ethanolamine phosphate)-glucosamine-myoinositol phosphate. Variations observed in additional mannose, N-acetylhexosamine, and ethanolamine phosphate moieties form this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)
We applied the improved sensitivity and soft ionization characteristics of electrospray Ionization (ESI)-MS/MS and matrix-assisted laser desorption/ionization(MALDI)-time of flight (TOF) mass spectrometry (MS) to analysis of the GPI-anchored C-terminal peptide derived from 5'-nucleotidase. ESI-MS/MS analysis was applied to the core structure (MW, 2,743). In the collision-induced dissociation (CID) spectrum, single-charged ions such as m/z 162 (glucosamine), 286 (mannose-phosphate-ethanolamine), and 447 ([mannose-phosphate-ethanolamine]-glucosamine) were clearly detected as characteristic fragment ions of the GPI-anchored peptide. On MALDI-TOF-MS analysis, heterogeneous peaks of GPI-anchored peptides were detected as single-charged ions in the positive mode. Product ions were obtained by post-source decay (PSD) of m/z 2,905 using curved field reflectron of TOF-MS. Most of the expected product ions derived from the GPI-anchored peptide, containing the core structure and an additional mannose side chain, were successively obtained. Thus, ESI-MS/MS and MALDI-TOF-PSD-MS proved to be effective and sensitive methods for analyzing the GPI-anchored peptide structure with less than 10 pmol of sample. These characteristic fragments or fragmentation patterns seem to be very useful for identification of GPI-anchored C-terminal peptides derived from any kind of GPI-anchored protein.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.